Malaria, particularly that caused by Plasmodium falciparum (Pf) and Plasmodium vivax (Pv), remains a global health concern. The rapid rise of drug resistance in malaria parasites has led to an urgent need to identify diverse, novel drug targets. Malarial parasites have a complex life cycle involving an asymptomatic ‘liver stage’ and a symptomatic ‘blood stage’. During the blood stage, the parasites utilise a proteolytic cascade to digest host haemoglobin, producing free amino acids necessary for parasite growth and reproduction. The final step of this cascade is catalysed by several metalloaminopeptidases, including a X-prolyl aminopeptidase P (PfAPP). The PfAPP has been shown to be essential for Pf growth suggesting it may be a suitable new drug target (Dalal and Klemba, 2007, Zhang et al., 2018). Previous work developed a chemical genetics platform to examine the substrate fingerprint of PfAPP and determined the crystal structure of PfAPP (Drinkwater et al., 2016). However, crystallography of the protein is not routine and remains challenging. To date, no potent inhibitors of PfAPP have been discovered and as such, drug validation of PfAPP remains unknown. In this study, the roles of the residues within the PfAPP S1 and S1’ pockets were dissected and the activity of homologous proteins from the other key human pathogen PvAPP and the murine malaria model Plasmodium berghei (PbAPP) was evaluated. The findings of this study provide critical insights into the mechanism of action of PfAPP, provide homologous proteins to potentially use as surrogates for structural biology techniques and offer a route to potent small molecule inhibitors of the enzyme family.